Galactosidase modified submucosal tissue

ABSTRACT

A tissue graft composition comprising submucosal tissue that has been enzymatically treated with galactosidase is described. The galactosidase modified submucosal tissue can be implanted to replace or support damaged or diseased tissues or utilized to form a cell culture growth substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national application of internationalapplication Ser. No. PCT/US98/18956 filed Sep. 11, 1998, which claimspriority to U.S. provisional application serial No. 60/058,545 filedSep. 11, 1997.

FIELD OF THE INVENTION

The present invention relates to enzymatically treated submucosal tissueand methods for its preparation and use. More particularly, the presentinvention is directed to submucosal tissue that is at least partiallydigested with galactosidase.

BACKGROUND OF THE INVENTION

It is known that compositions comprising the tunica submucosa of theintestine of warm-blooded vertebrates can be used advantageously astissue graft materials. See U.S. Pat. Nos. 4,902,508 and 5,281,422, thedisclosures of which are expressly incorporated herein by reference. Thetissue graft compositions described in those patents are characterizedby excellent mechanical properties, including high compliance, a highburst pressure point, and an effective porosity index which allows suchcompositions to be used beneficially for vascular graft and connectivetissue graft constructs. When used in such applications the graftconstructs appear not only to serve as a matrix for the regrowth of thetissues replaced by the graft constructs, but, indeed, to promote orinduce such regrowth of endogenous tissue. Common events to thisremodeling process include widespread and rapid neovascularization,proliferation of granulation mesenchymal cells,biodegradation/resorption of implanted intestinal submucosal tissuematerial, and lack of immune rejection.

It is also known that intestinal submucosa can be fluidized bycomminuting and/or enzymatic digestion, without loss of its apparentbiotropic properties, for use in less invasive methods of administration(e.g., by injection or topical application) to host tissues in need ofrepair. See U.S. Pat. No. 5,275,826, the disclosure of which isexpressly incorporated herein by reference.

Submucosal tissue grafts have been successfully used as a xenograft invascular, dura mater, urinary bladder, and orthopedic applications, andas dermal grafts. The remodeled tissue resembles the native tissue, bothgrossly and histologically, such that the original submucosal tissuegraft is generally unidentifiable when remodeling is complete. Despiteits xenogeneic nature, vertebrate submucosal tissue has not induced aclinical rejection response in the animal systems in which it has beentested, including rats, mice, dogs, cats, rabbits, and sheep. Thus,submucosal tissue is a potentially useful xenogeneic graft material foruse in humans.

Galactosyl-α(1,3)galactose (referred to as the Gal epitope) is aglycosyl modification of cell surface components and some serum proteinsin all mammals, except humans and Old World apes. The epitope comprisesa terminal galactose moiety linked to another galactose moiety throughan α1-3 linkage. It has been shown that human serum contains naturallyoccurring IgG and IgM antibodies directed against this epitope. It isestimated that 1% of all circulating IgG in humans is anti-Gal. Thishigh level of anti- Gal epitope antibodies is thought to be produced inresponse to endogenous bacteria in the gastrointestinal system; thelipopolysaccharides of those bacteria contain the Gal epitope.Xenogeneic transplantation of organ tissue into a human host results inIgG and IgM antibodies binding to the Gal epitope (especially for thoseepitopes located on endothelial cells), the initiation of aninflammatory reaction, and vascular thrombosis and hyperacute xenograftrejection of the transplant. Accordingly, a major obstacle to successfulxenotransplantation of porcine and other non-Old World ape vertebratespecies organs into humans is the presence of Gal epitopes on thetissues of those organs.

As disclosed herein, the Gal epitope has also been found in porcinesubmucosal tissue prepared in accordance with the procedures disclosedin U.S. Pat. Nos. 4,902,508 and 5,281,422. It is not known whether theGal epitope exists as a naturally occurring component of the submucosaltissue or whether the epitope is a remnant of cell lysis, and remainsattached to the submucosal tissue during processing of the submucosaltissue.

There have been no reports of submucosal tissue graft constructsinducing an immune response after implantation in the animal systems inwhich it has been tested, including rats, mice, dogs, cats, rabbits, andsheep. However, due to the association of the Gal epitope withhyperacute xenograft whole organ transplant rejection in humans, apreferred submucosal tissue graft construct would comprise submucosaltissue substantially free of the Gal epitope.

SUMMARY OF THE INVENTION

The present invention is directed to submucosal tissue that has beentreated with galactosidase to produce submucosal tissue substantiallyfree of detectable amounts of the Gal epitope. Such “Gal free”submucosal tissue is used to form tissue graft constructs for thereplacement and repair of damaged or diseased endogenous tissues.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions:

The term “Gal epitope” refers to a glycosyl modification[galactosyl-α(1,3)galactose] of cellular compounds present on the cellsurface or on serum proteins of all mammals except humans and Old Worldapes.

The term “glycosidase” as use herein refers to an enzyme thatcleaves/destroys the terminal a-linked galactose present on mostvertebrate cellular components. For example one glycosidase useful inaccordance with the present is α-galactosidase.

The term “glycosaminoglycanase” (GAGase) as use herein refers to anenzyme that hydrolyzes glycosaminoglycans (GAG) including, for examplechondroitin sulfate, hyaluronic acid heparin and heparin sulfate.

The term “Gal free submucosal tissue” refers to submucosal tissue thatis substantially free of all detectable amounts of the Gal epitope asdetermined by the antibody and lectin assays described in detail inExample 1.

The present invention is directed to warm blooded vertebrate submucosaltissue that is substantially free of the Gal epitope, and methods forits preparation and use. More particularly, the present invention isdirected to submucosal tissue that has been at least partially digestedwith an enzyme, such as α-galactosidase, to diminish the level of theGal epitope present in the submucosal tissue. The galactosidase treatedsubmucosal tissue is used in accordance with the present invention as anon-immunogenic tissue graft composition and as an in vitro cell culturesubstrate.

The galactosidase treated submucosal tissue of the present invention isderived from vertebrate submucosa and comprises naturally associatedextracellular matrix proteins, glycoproteins and other factors.Preferably the submucosal tissue comprises intestinal submucosa of awarm-blooded vertebrate, and one particularly preferred source of thesubmucosal tissue is the small intestine of warm-blooded vertebrates.Suitable submucosal tissue comprises the tunica submucosa delaminatedfrom the tunica muscularis and at least the luminal portion of thetunica mucosa. In one preferred embodiment of the present invention thesubmucosal tissue is intestinal submucosa comprising the tunicasubmucosa and basilar portions of the tunica mucosa including thelarnina muscularis mucosa and the stratum compactum which layers areknown to vary in thickness and in definition dependent on the sourcevertebrate species. Submucosal tissue can also be prepared from otherorgans of vertebrate species, for example, from the urogenital system,including the urinary bladder (see U.S. Pat. No. 5,554,389), and otherportions of the digestive tract including the stomach. The disclosuresof U.S. Pat. No. 5,554,389 is expressly incorporated herein.

The preparation of submucosal tissue for use in accordance with thisinvention is described in U.S. Pat. Nos. 4,902,508 and 5,554,389. Tosumrnmarize, submucosal tissue is prepared from vertebrate intestine (orother organ source), preferably harvested from porcine, ovine or bovinespecies, but not excluding other species, by subjecting the intestinaltissue to abrasion using a longitudinal wiping motion to remove theouter layers, comprising smooth muscle tissues, and the innermost layer,i.e., at least the luminal portion of the tunica mucosa. The submucosaltissue is rinsed with saline and optionally sterilized; it can be storedin a hydrated or dehydrated state. Lyophilized or air dried submucosaltissue can be rehydrated and used in accordance with this inventionwithout significant loss of its cell proliferative activity.

Stomach submucosa is prepared from a segment of stomach in a proceduresimilar to the preparation of intestinal submucosa. A segment of stomachtissue is first subjected to abrasion using a longitudinal wiping motionto remove the outer layers (particularly the smooth muscle layers) andthe luminal portions of the tunica mucosa layers. The resultingsubmucosa tissue has a thickness of about 100 to about 200 micrometers,and consists primarily (greater than 98%) of a cellular, eosinophilicstaining (H&E stain) extracellular matrix material.

The submucosal tissue specified for use in accordance with thisinvention can also be in a fluidized form. Submucosal tissue can befluidized by comminuting the tissue and optionally subjecting it toenzymatic digestion. The preparation of fluidized forms of submucosaltissue is described in U.S. Pat. No. 5,275,826, the disclosure of whichis expressly incorporated herein by reference. Fluidized forms ofsubmucosal tissue are prepared by comminuting submucosal tissue bytearing, cutting, grinding, or shearing the harvested submucosal tissue.In accordance with one embodiment, pieces of submucosal tissue arecomminuted by shearing in a high speed blender, or by grinding thesubmucosa in a frozen or freeze-dried state to produce a powder that canthereafter be hydrated with water or a buffered saline to form asubmucosal fluid of liquid, gel or paste-like consistency.

The native or fluidized submucosa formulation can be treated with anenzyme for a period of time sufficient to solubilize all or a majorportion of the submucosal tissue components. Preferably submucosaltissue is digested with an enzyme that hydrolyzes the structuralcomponents of the submucosal tissue to produce a suspension orhomogenous solution of submucosal tissue components. Submucosal tissuecan be enzymatically treated with proteases (for example, a collagenaseor trypsin or pepsin), glycosaminoglycanases or a combination ofproteases and glycosaminoglycanases. Optionally, other appropriateenzymes (i.e. those that hydrolyze the structural components of thesubmucosal tissue without substantially adversely impacting thebiotropic properties of the tissue) can be used alone or in combinationwith proteases and glycosaminoglycanases. The tissue digest can beoptionally filtered to provide a homogenous solution of partiallysolubilized submucosal tissue.

The viscosity of fluidized submucosa for use in accordance with thisinvention can be manipulated by controlling the concentration of thesubmucosa component and the degree of hydration. The viscosity can beadjusted to a range of about 2 to about 300,000 cps at 25° C. Higherviscosity formulations, for example, gels, can be prepared from thesubmucosa digest solutions by adjusting the pH of such solutions toabout 6.0 to about 7.0.

The present invention also contemplates the use of powder forms ofsubmucosal tissue. In one embodiment a powder form of submucosal tissueis prepared by pulverizing submucosal tissue under liquid nitrogen toproduce particles ranging in size from 0.1 to 1 mm². The particulatecomposition is then lyophilized overnight and sterilized to form a solidsubstantially anhydrous particulate composite. Alternatively, a powderform of submucosal tissue can be formed from fluidized submucosal tissueby drying the suspensions or solutions of comrninuted submucosal tissue.

It is anticipated that each of the various forms of submucosal tissue(native, fluidized, protease or GAGase treated and powder forms) has theGal epitope associated with the tissue. In accordance with the presentinvention, those various forms of submucosal tissue can be furthermodified to reduce the amount of Gal epitope present in the tissue.Alternatively, the native submucosal tissue can be first enzymaticallytreated to reduce the amount of Gal epitope present in the tissue beforethe tissue is fluidized, protease or GAGase treated, or formed intopowder form. In one embodiment, native submucosal tissue is treated withα-galactosidase to produce a Gal epitope depleted submucosal tissue. Ina preferred embodiment the submucosal tissue is hydrolyzed withα-galactosidase until it is substantially free of detectable amounts ofthe Gal epitope. The Gal free tissue can then optionally be furthermanipulated to produce the described fluidized, protease/GAGase treated,and powder forms of submucosal tissue.

As the experimental data of Example 1 indicate, the Gal epitope isassociated with porcine submucosal tissue. The Gal epitope was detectedin porcine submucosal tissue by immunohistochemical staining methodsusing both a naturally occurring lectin (IB-4) that binds to the Galepitope and freshly collected pooled human serum as the source ofprimary antibodies. Pretreating the porcine submucosal tissue with humanserum decreased the intensity of staining by the IB4 lectin, andpretreating the tissue with the IB-4 lectin decreased the intensity ofantibody staining. Accordingly, both the IB-4 lectin and the pooledprimary antibodies recognized the same site. α-Galactosidase treatmentof the submucosal tissue effectively reduced staining with the lectin tonegligible amounts, indicating that the Gal epitope could be cleavedfrom the submucosal tissue by treatment with α-galactosidase.

It is unknown whether the epitope exists as a naturally occurringcomponent of the submucosal tissue extra cellular matrix or whether theepitope is a remnant of cell lysis and remains attached to thesubmucosal tissue extra cellular matrix during processing of thesubmucosal tissue. Since the Gal epitope has been detected on serumproteins, and thus is not exclusive to cell surfaces, the Gal epitopemay be present on extracellular matrix proteins that comprise submucosaltissue. Additionally, since submucosal tissue preparation begins withmechanical delamination of the jejunum, which certainly results in therupture of a significant number of cells, cellular components depositedduring the preparation of submucosal tissue may also contribute to thepresence of the epitope in the submucosal tissue.

As noted above the presence of the Gal epitope on nonhuman mammaliantissues has been an obstacle to xenograft transplantations due tohyperacute rejection of the xenograft. The association of the Galepitope with porcine intestinal submucosal tissue raises the issue ofwhether submucosal tissue could activate complement in fresh humanplasma thus leading to rejection or complement mediated destruction ofthe tissue upon implantation into humans. Hyperacute rejection resultsfrom complement activation, especially in the blood supply to thegrafted organ. The complement system is a complex system ofapproximately 20 interacting components and is involved inantibody-mediated cell lysis of cells as well as stimulating phagocyticcells to ingest and destroy cells. Most of the components of thecomplement system are in an inactive form and are activated by asequence of proteolytic activation reactions triggered by an immuneresponse. In particular the complement cascade can be activated byantibody-antigen reaction, such as that between an anti-Gal antibody andthe Gal epitope.

The pivotal component of the complement system proteolytic cascade isC3. C3 can be activated by two different pathways, the classical pathwayand the alternative pathway, in both cases C3 is cleaved by an enzymecomplex into C3a and C3b. C3b continues the cascade and C3a is involvedin anaphylaxis, chemotaxes, and acute inflammation. A radioimmunoassaykit to measure C3a is commercially available (Amersham) and can beutilized as an indicator of complement activation. This kit was used todetermine if submucosal tissue induces complement activation in humanplasma (See Example 2). Based on the results of this assay, sheets ofporcine submucosal tissue prepared in accordance with the presentinvention do not activate complement, despite the presence ofimmunologically detectable Gal epitopes.

The fact that submucosal tissue does not stimulate complement activationis surprising, considering the strongly positive staining observed withthe HB-4 lectin and human serum. This suggests that naturally occurringhuman IgG and IgM bind to the submucosal tissue, but fail to fixcomplement significantly in vitro. Complement activation requiresbinding of IgG or IgM to the antigen in relatively high density.Accordingly, the lack of iii vitro complement response may result from alow density of the Gal epitopes in the tissue. Furthermore, IgG/antigencomplexes activate complement less efficiently than IgM/antigencomplexes, especially at low antigen densities, and the competition ofIgG for binding with the Gal epitope may also contribute to the lowcomplement response. Finally, the process of cryosectioning and fixingmay expose more antigenic sites for immunoperoxidase labeling than areavailable in the native tissue, resulting in impressive immunoperoxidasestaining despite the poor complement activation. In particular the poresize and distribution of the pores in the native submucosal tissue mayrestrict complement access to only the most superficial structures,effectively decreasing the amount of antigen available for binding tothe antigen.

Regardless of porcine submucosal tissue's ability to activate complementin vitro upon exposure to human serum, due to the association of the Galepitope with hyperacute xenograft whole organ transplant rejection inhumans, a preferred graft construct would comprise a tissuesubstantially free of the Gal epitope. Gal free submucosal tissue can beprepared in accordance with the present invention by contacting thesubmucosal tissue with an enzymatic solution wherein the enzyme destroysor separates the Gal epitope from the submucosal tissue. Preferably theenzyme is α-galactosidase.

Submucosal tissue treated to remove the Gal epitope can be in its nativestate, or in a fluidized, suspension, solution or powdered forms. Thetissue is contacted with the enzyme under conditions (includingtemperature, pH, salt concentration, etc) suitable for enzymaticactivity. The digestion is conducted for a time sufficient to reduce theGal epitope content of the tissue. Preferably, the Gal epitopeconcentration associated with the tissue is reduced by greater than 50%,more preferably Gal epitope concentration is reduced by greater than 90%and in accordance with one embodiment vertebrate submucosal tissue isenzymatically treated to be substantially free of detectable amounts ofthe Gal epitope. After the tissue has been enzymatically digested todeplete the Gal epitope content, the tissue is repeatedly washed insaline or a suitable buffered solution to remove the cleaved epitope andthe enzyme. Alternatively, after enzymatic digestion to deplete Galepitope content, the tissue can be dialyzed against a buffered solutionto remove the cleaved epitope and enzyme.

The length of time the submucosal tissue is digested with the enzyme isdependent on the amount of tissue to be treated relative to theconcentration of the enzyme (assuming environmental factors such astemperature, pH, salt concentration, etc have been optimized forenzymatic activity). One preferred group of enzymes utilized fordepleting the Gal epitope content of submucosal tissue includesgalactosidases, including particularly the use of α-galactosidase,either alone or in combination with other galactosidases or otherenzymes.

In accordance with one embodiment the submucosal tissue is treated withα-galactosidase at a concentration ranging from about 5 to about 100units/ml, and more preferably about 10 to about 50 units/ml for 6-12hours. Each digestion reaction typically comprises approximately about10 to about 100 mg of submucosal tissue, and more preferably about 40 toabout 60 mg of submucosal tissue. Accordingly, about 0.2 to about 5units of enzyme are added per 1 mg of submucosal tissue, and morepreferably about 0.25 to about 2 units of enzyme are added per 1 mg ofsubmucosal tissue and the tissue is incubated at 37° C. for 6-12 hours.In one embodiment, 20 units/ml of α-galactosidase are added to thesubmucosal tissue and the tissue is digested for 8 to 10 hours at 37° C.The submucosal tissue can be fluidized or partially dehydrated prior tocontact with the solution containing one or more galactosidases toassist enzymatic digestion of the Gal epitopes.

In one embodiment a Gal free tissue graft construct comprisingintestinal submucosa delaminated from the tunica muscularis and at leastthe luminal portion of the tunica mucosa of vertebrate intestine isprepared by contacting the tissue with galactosidase for a sufficientamount of time to remove substantially all of the Gal epitope. Forlarger pieces of submucosal tissue, the tissue can be dehydrated orperforated before being contacted with the galactosidase solution toenhance access of the enzyme to the Gal epitope present in the tissueand decrease the time of digestion. In another embodiment fluidizedforms of the Gal free submucosal tissue (including enzymaticallydigested suspensions and solutions of submucosal tissue) are treated toremove the Gal epitope and are then gelled to form a solid or semi-solidmatrix.

Enzymatic digestion of the submucosal tissue to remove the Gal epitopecan be accomplished without loss of the biotropic properties of thenative submucosal tissue. It has been previously reported thatsubmucosal tissue can be utilized as a cell culture substrate (see U.S.Pat. No. 5,695,998, the disclosure of which is expressly incorporatedherein). As demonstrated by the data of Example 3, Gal free submucosaltissue exhibits similar properties as a cell culture substrate, withregards to cell proliferation and differentiation its vitro, as nativesubmucosal tissue. Accordingly, the removal of the Gal epitope does notappear to affect submucosal tissue's ability to stimulate the growth anddifferentiation of cells, and therefore, the Gal free submucosal tissuecan be utilized in tissue graft compositions to stimulate the repair ofdamaged or diseased tissues.

In accordance with the present invention, the Gal free submucosal tissuecompositions of the present invention are used advantageously to inducethe formation of endogenous tissue at a desired site in a warm bloodedvertebrate. The method comprises the step of contacting the damaged ordiseased site with a graft composition comprising Gal free submucosaltissue in an amount effective to induce endogenous tissue growth at thesite the composition is administered. The Gal free submucosal tissuecompositions can be administered to the host in either solid or sheetform, by surgical implantation, or in fluidized form, by injection.

In particular, the Gal free submucosal tissue compositions of thepresent invention lend themselves to a wide variety of surgicalapplications relating to the repair or replacement of damaged tissues,including, for example the repair of vascular and connective tissues.Connective tissues for the purposes of the present invention includebone, cartilage, muscle, tendons, ligaments, and fibrous tissueincluding the dermal layer of skin. The use of the Gal free submucosaltissue segments in the repair or replacement of connective tissues canbe conducted using the same procedures described in U.S. Pat. Nos.5,281,422 and 5,352,463, expressly incorporated herein by reference.Furthermore, it is anticipated that Gal free submucosal tissue graftconstructs will have utility in the replacement and repair of vascular,neural, dura mater, urinary bladder, and dermal tissues.

The present Gal free submucosal tissue composition may be sterilizedusing conventional sterilization techniques including tanning withglutaraldehyde, formaldehyde tanning at acidic pH, ethylene oxidetreatment, propylene oxide treatment, gas plasma sterilization, gammaradiation, and peracetic acid sterilization. A sterilization techniquewhich does not significantly weaken the mechanical strength andbiotropic properties of the graft is preferably used. For instance, itis believed that strong gamma radiation may cause loss of strength inthe graft material. Because one of the most attractive the presentintestinal submucosa grafts is their ability to induce host-remodelingresponses, it is desirable not to use a sterilization approach whichwill detract from that property. Preferred sterilization techniquesinclude exposing the graft to peracetic acid, low dose gamma irradiationand gas plasma sterilization; peracetic acid sterilization being themost preferred method. Typically, after the tissue graft composition hasbeen sterilized, the composition is wrapped in a porous plastic wrap andsterilized again using electron beam or gamma irradiation sterilizationtechniques.

Gal free submucosal tissue can also be used in accordance with thisinvention as a cell growth substrate in a variety of forms, includingits native sheet-like configuration, as a gel matrix, as a supplementalcomponent in art-recognized cell/tissue culture media, or as coating forculture-ware to provide a more physiologically relevant substrate thatsupports and enhances the proliferation of cells in contact with thesubmucosal matrix. The composition comprises submucosal tissue that hasbeen fluidized by enzymatically digesting the tissue with an enzymeselected from the group consisting essentially of proteases and GAGasesand gelling the digested tissue by adjusting the pH of the submucosaltissue to about 6.0 to about 7.0. Typically the submucosal tissue issterilized prior to use in cell culture applications.

In one preferred embodiment, a composition comprising fluidizedvertebrate submucosa that has been treated with galactosidase to depletethe detectable levels of the Gal epitope is prepared by first treatingthe native submucosal tissue with galactosidase and then fluidizing thetissue. In particular, the submucosal tissue is prepared, in its nativesheet-like configuration, and contacted with a solution containinggalactosidase. The digestion is conducted for a time sufficient toreduce the Gal epitope content of the tissue, and preferably thesubmucosal tissue is enzymatically treated with galactosidase for asufficient time to be substantially free of detectable amounts of theGal epitope. After the tissue has been enzymatically digested to depletethe Gal epitope content, the tissue is repeatedly washed in saline or asuitable buffered solution to remove the cleaved epitope and the enzyme.The tissue is then fluidized by comminuting and/or enzymatic digestionas described above, and in U.S. Pat. No. 5,275,826.

EXAMPLE 1

The Presence of the Gal epitope in porcine submucosal tissue and itsremoval by enzymatic digestion.

Materials and Methods

Reagenits: Normal human serum (NHS) was prepared fresh from 4individuals and pooled. Human serum albumin was purchased fromCalbiochem. Goat anti-human IgG (Fc fragment) and antihuman IgM (μ chainspecific) conjugated to horseradish peroxidase (HRP) were purchased fromBethyl Labs (Montgomery, Tex.). The lectin I-B4 (from Griffontiasimplicifolia) HRP conjugate, α-galactosidase, and lipopolysaccharidefrom E. coli were purchased from Sigma (St. Louis, Mo.).Diaminobenzidine (DAB), the peroxidase substrate, was purchased fromVector Labs (Burlingame, Calif.). Complement C3a des-Argradioimmunoassay kit was purchased from Amersham (Madison, Wis.).Phosphate buffered saline (PBS), pH 7.4 was prepared as needed.

Tissues: Six hydrated submucosal tissue specimens from six differentpigs were prepared essentially as described in U.S. Pat. No. 4,902,508,and were supplied by Cook Biotech Inc. Fresh porcine liver was used as apositive control tissue. Samples were frozen in O.C.T.® embedding medium(Miles) as a cryoprotectant and stored at −70° C. until use.Cross-sectional slices of submucosal tissue (perpendicular to the tissuelong axis) were cut on a cryomicrotome set to a thickness of 7 μm andmounted on poly-L-lysine coated slides.

Immunohistochemical staining: Slides were allowed to warm to roomtemperature (RT) and fixed in acetone for 3 min at 4° C. Nonspecificprotein binding was blocked by incubating the slides in 2% human serumalbumin for 20 min. Endogenous peroxidase activity was inhibited byincubating for 30 min in 0.45% (v/v) H₂O₂ in methanol. The slides wererinsed in PBS for 10 min between incubations. All incubations wereperformed at RT except as noted.

The conditions used to stain the prepared tissues for the Gal epitopeare summarized in Table 1:

TABLE 1 Condition Block 1° Stain 2° Stain 1 none I-B₄-HRP none 2 noneNHS anti-human IgG- HRP 3 none NHS anti-human IgM- HRP 4 NHS I-B₄-HRPnone 5 I-B₄-HRP NHS anti-human IgG- HRP 6 I-B₄-HRP NHS anti-human IgM-HRP 7 α- I-B₄-HRP none galactosidase

Conditions 1-3 are simple immunoperoxidase staining procedures. Directstaining with I-B₄, which specifically binds Galα (1,3)Gal, was utilizedbecause no anti-lectin reagent was available. Under conditions 4-6 thetissue was first exposed to either the lectin-BRP or normal human serum,then followed through the normal staining procedure. In particular,tissues were blocked by incubation with lectin-HRP for 18 hr at RT, orserum for 1 hour at 37° C. followed by 18 hour at RT. In the case oflectin-HP blocking, the tissue was exposed to H₂O₂/methanol solutionafter lectin binding to inactivate both the endogenous peroxidases andthe HRP bound to the lectin.

The blocked tissues were then incubated in serum for 1 hour at 37° C.followed by 18 hours at room temperature (condition 5 and 6), or lectinfor 1 hour at room temperature, respectively (condition 4).Serum-stained tissues were incubated with anti-IgM or anti-IgG HPconjugate at RT for 1 hour (condition 2,3,5,6). For condition 7,α-galactosidase, which cleaves terminal α-galactosyl residues, wasemployed at 20 units/ml prior to lectin staining, the enzyme digestedtissues were then incubated for 18 hr at RT. All tissues were incubatedin peroxidase substrate (DAB) solution for 10 minutes and counterstainedwith hematoxylin for 10 seconds. Finally, the slides were dehydratedthrough a graded alcohol series and mounted with a coverslip. Positivestaining appears black against a purple background.

Results

Ali submucosal tissue samples stained strongly positive for the Galepitope with the I-B4 lectin and with human serum containing naturallyoccurring antibodies to the epitope. The epitope appears to betransmurally distributed throughout the submucosal tissue. The hepaticportal triads, which contain abundant endothelial cells, also stainedstrongly positive while the hepatocytes were moderately positive.Staining intensity was decreased with serum treatment prior tolectin-HRP, but was not entirely eliminated. These results indicate thatthe Gal epitopes were being at least partially masked by specificinteraction with the serum components.

Submucosal tissue treated with serum and stained with anti-IgG appearedsimilar to the lectin-stained tissue, while blocking with lectin-HRPsignificantly decreased staining intensity. Submucosal tissue treatedwith serum and stained with anti-IgM appeared similar to the anti-IgGstained tissue.

Digestion with α-galactosidase significantly decreased staining of bothsubmucosal tissue and liver by the I-B4 lectin (see α-Gal.+Lectin, Table2).

A summary of the results is presented in Table 2:

TABLE 2 Different Degree of Immunohistochemical Staining on CBI SISLectin + Lectin + Anti- Anti- Anti- Anti- body + body + α- body bodyAnti- Condit Lecti Anti- Anti- Gal. + Anti- Anti- body + No n IgG IgMLectin IgG IgM Lectin 1 +++ +++ +++ + ++ ++ ++ 2 +++ +++ +++ − ++ ++ ++3 +++ ++++ +++ + + ++ + 4 +++ ++++ +++ + ++ ++ ++ 5 +++ ++++ +++ + ++ +++ 6 +++ ++++ ++++ + ++ ++ ++ Staining recorded as − = absent, + = weak,++ to ++++ = increasing intensity

EXAMPLE 2

Submucosal Tissue's ability to Activate Complement

Complement activation assay: Plasma samples were collected from healthylaboratory staff The blood was drawn into EDTA (K₃) Venoject® tubes(Terumo Medical, Elkton, Md.) and centrifuged immediately at 1,500×g for15 min at 4° C. The plasma was removed from the cells and usedimmediately.

50 mg pieces of hydrated porcine submucosal tissue (blotted to removeexcess liquid) were incubated with 250 μl of plasma (normal orheat-inactivated) for 1 hour at 37° C. Samples from three different lotswere prepared in duplicate. The plasma was then separated from thesubmucosal tissue and assayed in accordance with the manufacturer'sinstructions for human complement C3a. Each sample was run intriplicate. In addition, 80 μl of a 10 mg/ml solution oflipopolysaccharide from E coli (purchased from Sigma) was also incubatedwith 250 μl of plasma (3.2 mg/ml of plasma) as a positive control.Controls also consisted of plasma incubated without submucosal tissue atboth RT, and at 37° C. for 1 hour.

Each lot of submucosal tissue was tested twice. Every sample was run intriplicate. Calculations were done in accordance with the manufacturer'sinstructions. The log concentration of the standards was plotted againstthe percent bound with Excel software. An exponential line equation wasdetermined and the concentration of C3 a in each of the triplicates wasdetermined. After taking into consideration the dilution step of theassay, the results for each sample were averaged and the standarddeviation determined. The results presented are from one experiment;however, the assay was performed 5 times with different pools of plasmaand the results were consistent. The results were compared forstatistical significance with single factor ANOVA.

Results plasma incubated at room temperature for 1 hour 154 ng/ml ± 13plasma incubated at 37° C. for 1 hour 332 ng/ml ± 134 plasma withlipopolysaccharide >16,000 ng/ml plasma with submucosal tissue lot 1,sample A 191 ng/ml ± 9 plasma with submucosal tissue lot 1, sample B 354ng/ml ± 170 plasma with submucosal tissue lot 2, sample A 234 ng/ml ± 14plasma with submucosal tissue lot 2, sample B 249 ng/ml ± 113 plasmawith submucosal tissue lot 3, sample A 220 ng/ml ± 50 plasma withsubmucosal tissue lot 3, sample B 393 ng/ml ± 144

Average and standard deviation of all 6 tubes of:

submucosal tissue 1 273 ng/ml ± 140 submucosal tissue 2 241 ng/ml ± 72submucosal tissue 3 307 ng/ml ± 135

Average and standard deviation of all 18 tubes of submucosal tissue:

274 ng/ml±116

The results for the complement C3a assay, shown above, indicate thatintestinal submucosa prepared in accordance with the proceduresdescribed in U.S. Pat. No. 4,902,508 (i.e., without treatment to removethe Gal epitope) does not produced significant complement activation invitro. Neither of the negative control plasma samples producedsignificant complement activation in vitro, nor did the submucosaltissue treated samples, the differences between these three samples werenot significantly different (p=0.15). The lipopolysaccharide (positivecontrol) showed complement C3a concentration greater than 16,000 ng/ml,which was off the standard curve.

EXAMPLE 3

The use of Galα(1,3)Gal epitope depleted submucosal tissue as a cellgrowth substrate.

Material and Methods

Cell lines

Fetal rat keratinocytes (FR)

Swiss 3T3 fibroblast cells (M)

Procedure

Hydrated submucosal tissue in sheet form was supplied by Cook BiotechInc. The submucosal tissue was placed in a plastic holder to keep thetissue flat and ensure good contact of the cells with the tissue. Thetissue was treated with α-galactosidase to remove the Gal epitope asfollows: α-galactosidase was suspended in 3.5 M ammonium sulfate, 50 mMsodium acetate, pH 5.5 to a final concentration of 20 units/ml. Thetreatment solution (50 μl) was added to the well of submucosal tissueholder, and the submucosal tissue (50 mg) was incubated at roomtemperature overnight. Three samples of submucosal tissue were treatedunder the following conditions:

1. submucosal tissue incubated with α-galactosidase

2. submucosal tissue incubated with 3.5 M ammonium sulfate, 50 mM sodiumacetate, pH 5.5

3. submucosal tissue incubated with water

After incubating the tissues overnight, the tissue samples were rinsedwith PBS followed by complete cell culture medium. A submucosal tissueholder was placed in each well of a 12-well plate, and 2 ml of completeculture medium was added to the well. 20,000 FR cells or 13,000 3T3cells were seeded onto the submucosal side of the submucosal tissue in atotal volume of 250 μl. The plate was placed in a 37° C. CO₂ incubator.At 48 hours and 1 week, samples were fixed in neutral buffered formalinfor histologic evaluation (hematoxylin and eosin stained), or 2%glutaraldehyde in phosphate buffered saline for examination by scanningelectron microscopy.

Results

FR cells grew and differentiated similarly on all submucosal tissuesamples. There were more cells present at 1 week than after 48 hours.There were no significant differences observed between the three treatedsubmucosal tissue substrates.

3T3 cells also grew and differentiated similarly on all 3 submucosaltissue substrates. There were more cells present at 1 week than after 48hours. There were no significant differences observed between threetreated submucosal tissue substrates.

What is claimed is:
 1. A tissue graft composition comprising vertebratesubmucosal tissue enzymatically treated to be substantially free of theGal epitope.
 2. The tissue graft composition of claim 1 wherein thesubmucosal tissue is a segment of intestinal submucosa comprising thetunica submucosa delamninated from the tunica muscularis and at leastthe luminal portion of the tunica mucosa of vertebrate intestine.
 3. Thetissue graft composition of claim 1 wherein the submucosal tissue istreated with galactosidase.
 4. The tissue graft composition of claim 3,wherein the submucosal tissue is digested with an enzyme for a period oftime sufficient to solubilize the tissue.
 5. The tissue graftcomposition of claim 4 gelled to form a solid or semi-solid matrixsuitable for culturing eukaryotic cells on the surface of the matrix. 6.A tissue graft construct comprising intestinal submucosa delaminatedfrom the tunica muscularis and at least the luminal portion of thetunica mucosa of vertebrate intestine and incubated with galactosidasefor a sufficient amount of time to be substantially free of the Galepitope.
 7. A composition for supporting the growth of a cellpopulation, said composition comprising submucosal tissue that has beenfluidized by enzymatically digesting the tissue with an enzyme selectedfrom the group consisting essentially of proteases and GAGases;enzymatically treated with galactosidase to remove substantially alldetectable amounts of the Gal epitope; and gelled by adjusting the pH ofa solution of enzymatically digested submucosal tissue to about 6.0 toabout 7.0.